The present invention relates to methods and apparatus for filtering a fluid, and more particularly relates to the use of centrifugal force to aid such filtration.
It is well known that the filtration of fluids by microporous and ultraporous filters may be carried out under the influence of centrifugal force. For example, it is often desired to isolate proteins from liquid samples of bodily fluids or biological growth media in order either to concentrate the proteins or to produce a protein-free filtrate. U.S. Pat. No. 3,488,768 (P.N. Rigopulos, 1970) discloses several apparatus and methods for performing such filtration. Rigopulos also teaches that in addition to providing the trans-membrane force necessary to move the liquid through the filter, centrifugal force can be used to maintain the working surface of the filter free from a clogging build-up of protein or sediment.
U.S. Pat. No. 3,960,727 (Hochstrasser) discloses methods and apparatus for separating blood serum from clotted whole blood, preferably aided by centrifugal force. A sample to be filtered is contained in a first tube and a second tube, including a filter at its lower end, is placed therein. The second tube slowly sinks down into the first tube and through the sample, filtering the sample as it sinks. Sinking is advantageously accelerated by subjecting the assembly to centrifugal force by spinning the assembly in a centrifuge.
We have found that during the filtration of rather dilute solutions, such as dilute solutions of proteins or polypeptides, the "full floating" inner filtration tube design of Hochstrasser does not provide optimal trans-membrane pressure during centrifugation. Other problems occur in other devices during the filtration of highly concentrated solutions or solutions containing a large volume of particulate matter, as the filter may rapidly become clogged by the solute or particles. A need remains for apparatus and methods of performing centrifugal force-enhanced filtration of fluids.
An object of the present invention is to provide filtration methods and apparatus useful for both dilute solutions and particle-laden samples.
Another object of the present invention is to provide such an apparatus which, when placed within a 50 ml centrifuge tube carrier, is capable of filtering a 5 to 15 ml volume of sample.
A further object of this invention is to provide complete containment of biohazardous samples during centrifugal force-enhanced filtration.
The present invention satisfies these needs by providing methods and apparatus for filtering a fluid, including both relatively dilute solutions of e.g. protein(s) or polypeptide(s) and relatively particulate samples such as fermentation broth containing cells or whole blood. The apparatus consists of a container for holding a sample of liquid to be filtered, the container having an open end and a closed end. A filtering vessel is installed within the container and includes a chamber for receiving filtered fluid. The filtering vessel is preferably cylindrical and has an open upper end and an opening at its lower end which is covered by a filter, preferably an ultrafiltration (i.e. pore size of less than about 100 Angstroms) membrane carried by a filter support. High trans-membrane pressure and filtration rates are obtained by forcibly immersing the end of the filtering vessel which carries the filter into the sample to create a pressure head above the filter. By "forcibly immersing" we mean immersing against the natural buoyancy of the filtering vessel. The immersed position of the filtering vessel must be maintained, and this is accomplished in our preferred embodiment by the combination of an annular locking cap and the engagement of a ridge on the filtration vessel with the inner edge of the annular cap. Although the pressure differential created by forcible immersion alone may not be sufficient to accomplish filtration, this pressure differential is greatly increased when, according to the inventive method, the apparatus is subjected to centrifugal force. The centrifugal force greatly amplifies the pressure differential between the inside and outside of the filtration vessel to force the sample liquid through the filter and into the filtration vessel. The centrifugal force simultaneously prevents the working side of the filter from becoming clogged with sediment. Liquid within the filtration vessel is known as "filtrate" while that portion of the sample remaining outside the filtration vessel is known as "retentate".